Hyeonjin Choi

Reactive Oxygen Species Facilitate Adipocyte Differentiation by Accelerating Mitotic Clonal Expansion

Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-β (C/EBPβ) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBPβ is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBPβ is sequentially phosphorylated by MAPK and glycogen synthase kinase-3β, inducing C/EBPβ DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBPβ is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBPβ DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H2O2 and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor γ. Interestingly, cell cycle progression (S to G2/M phase) was markedly enhanced by H2O2, whereas antioxidants caused an S phase arrest during the MCE. H2O2 treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBPβ, which is a hallmark for C/EBPβ binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBPβ. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBPβ DNA binding activity, along with the dual phosphorylation of C/EBPβ, is required for the MCE and terminal differentiation of adipocytes.

Nuclear receptor PPARγ-regulated monoacylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipid accumulation in diet-induced hepatic steatosis.

Recently, hepatic peroxisome proliferator-activated receptor (PPAR)γ has been implicated in hepatic lipid accumulation. We found that the C3H mouse strain does not express PPARγ in the liver and, when subject to a high-fat diet, is resistant to hepatic steatosis, compared with C57BL/6 (B6) mice. Adenoviral PPARγ2 injection into B6 and C3H mice caused hepatic steatosis, and microarray analysis demonstrated that hepatic PPARγ2 expression is associated with genes involved in fatty acid transport and the triglyceride synthesis pathway. In particular, hepatic PPARγ2 expression significantly increased the expression of monoacylglycerol O-acyltransferase 1 (MGAT1). Promoter analysis by luciferase assay and electrophoretic mobility shift assay as well as chromatin immunoprecipitation assay revealed that PPARγ2 directly regulates the MGAT1 promoter activity. The MGAT1 overexpression in cultured hepatocytes enhanced triglyceride synthesis without an increase of PPARγ expression. Importantly, knockdown of MGAT1 in the liver significantly reduced hepatic steatosis in 12-wk-old high-fat–fed mice as well as ob/ob mice, accompanied by weight loss and improved glucose tolerance. These results suggest that the MGAT1 pathway induced by hepatic PPARγ is critically important in the development of hepatic steatosis during diet-induced obesity.

Caffeine inhibits adipogenesis through modulation of mitotic clonal expansion and the AKT/GSK3 pathway in 3T3-L1 adipocytes

Caffeine has been proposed to have several beneficial effects on obesity and its related metabolic diseases; however, how caffeine affects adipocyte differentiation has not been elucidated. In this study, we demonstrated that caffeine suppressed 3T3-L1 adipocyte differentiation and inhibited the expression of CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ, two main adipogenic transcription factors. Anti-adipogenic markers, such as preadipocyte secreted factor (Pref)-1 and Krüppel-like factor 2, remained to be expressed in the presence of caffeine. Furthermore, 3T3-L1 cells failed to undergo typical mitotic clonal expansion in the presence of caffeine. Investigation of hormonal signaling revealed that caffeine inhibited the activation of AKT and glycogen synthase kinase (GSK) 3 in a dose-dependent manner, but not extracellular signal-regulated kinase (ERK). Our data show that caffeine is an anti-adipogenic bioactive compound involved in the modulation of mitotic clonal expansion during adipocyte differentiation through the AKT/GSK3 pathway. [BMB Reports 2016; 49(2): 111-115]

Dexras1 mediates glucocorticoid-associated adipogenesis and diet-induced obesity

Significance Glucocorticoids are well known to play a major role in obesity, but underlying mechanisms have been obscure. We demonstrate that the small G protein Dexras1, first identified based on its dramatic induction by glucocorticoids, mediates adipogenic differentiation of preadipocytes, as well as diet-induced obesity in intact rodents. Thus, the adipogenesis of preadipocytes is abolished by Dexras1 deletion and selectively induced by Dexras1 expression. Relevance to intact animals is evident from our experiments wherein diet-induced obesity is prevented in mice with knockout of Dexras1. Thus, pharmacotherapy involving Dexras1 may afford a promising approach to the therapy of obesity. Adipogenesis, the conversion of precursor cells into adipocytes, is associated with obesity and is mediated by glucocorticoids acting via hitherto poorly characterized mechanisms. Dexras1 is a small G protein of the Ras family discovered on the basis of its marked induction by the synthetic glucocorticoid dexamethasone. We show that Dexras1 mediates adipogenesis and diet-induced obesity. Adipogenic differentiation of 3T3-L1 cells is abolished with Dexras1 depletion, whereas overexpression of Dexras1 elicits adipogenesis. Adipogenesis is markedly reduced in mouse embryonic fibroblasts from Dexras1-deleted mice, whereas adiposity and diet-induced weight gain are diminished in the mutant mice.

Krüppel-like factor KLF8 plays a critical role in adipocyte differentiation.

KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPARγ2 and C/EBPα promoter activity, suggesting that KLF8 is an upstream regulator of PPARγ and C/EBPα. The KLF8 binding sites were localized by site mutation analysis to −191 region in C/EBPα promoter and −303 region in PPARγ promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.

O-GlcNAc modification is essential for the regulation of autophagy in Drosophila melanogaster

O-GlcNAcylation is a dynamic post-translational modification that takes place on ser/thr residues of nucleocytoplasmic proteins. O-GlcNAcylation regulates almost all cellular events as a nutrient sensor, a transcriptional and translational regulator, and a disease-related factor. Although the role of O-GlcNAcylation in insulin signaling and metabolism are well established, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we manipulated O-GlcNAcylation in Drosophila and found that it regulates autophagy through Akt/dFOXO signaling. We demonstrate that O-GlcNAcylation and the levels of O-GlcNAc transferase (OGT) are increased during starvation. Furthermore, Atg proteins and autolysosomes are increased in OGT-reduced flies without fasting. Atg proteins and autophagosomes are reduced in OGT-overexpressing flies. Our results suggest that not only autophagy gene expression but also autophagic structures are regulated by OGT through Akt and dFOXO. These data imply that O-GlcNAcylation is important in modulating autophagy as well as insulin signaling in Drosophila.

c-Jun regulates adipocyte differentiation via the KLF15-mediated mode

Abnormal adipocyte differentiation is implicated in the development of metabolic disorders such as obesity and type II diabetes. Thus, an in-depth understanding of the molecular mechanisms associated with adipocyte differentiation is the first step in overcoming obesity and its related metabolic diseases. Here, we examined the role of c-Jun as a transcription factor in adipocyte differentiation. c-Jun overexpression in murine 3T3-L1 preadipocytes significantly inhibited adipocyte differentiation. In addition, the expression level of KLF15, an upstream effector of the key adipogenic factors C/EBPα and PPARγ, was decreased upon the ectopic expression of c-Jun. We found that c-Jun inhibited basal and glucocorticoid receptor (GR)-induced promoter activities of KLF15. c-Jun directly bound near the glucocorticoid response element (GRE) sites in the KLF15 promoter and inhibited adjacent promoter occupancies of GR. Furthermore, the restoration of KLF15 expression in 3T3-L1 cells with the stable ectopic expression of c-Jun partially rescued adipocyte differentiation. Our results demonstrate that c-Jun can suppress adipocyte differentiation through the down-regulation of KLF15 at the transcriptional level. This study proposes a novel mechanism by which c-Jun regulates adipocyte differentiation.

Radiofrequency thyroarytenoid myothermy for treatment of adductor spasmodic dysphonia: how we do it

Dear Editor, Adductor spasmodic dysphonia is a neurological voice disorder characterised by action-induced, task-specific spasm of the vocal folds. During the past decade, botulinum toxin has emerged as an accepted treatment for adductor spasmodic dysphonia. However, this treatment has some limitations and therefore a variety of surgical therapies for adductor spasmodic dysphonia have been proposed. Recently, Remacle reported a promising outcome with transoral bipolar radiofrequency (RF)-induced thermotherapy by coagulating terminal branches of the recurrent laryngeal nerve in three patients with adductor spasmodic dysphonia. We performed a clinical trial of patients with adductor spasmodic dysphonia with a modified Remacle’s procedure. Instead of coagulating the distal part of one recurrent laryngeal nerve, we ablated portions of the thyroarytenoid muscles.

O-GlcNAcylation of eIF2α regulates the phospho-eIF2α-mediated ER stress response.

O-GlcNAcylation is highly involved in cellular stress responses including the endoplasmic reticulum (ER) stress response. For example, glucosamine-induced flux through the hexosamine biosynthetic pathway can promote ER stress and ER stress inducers can change the total cellular level of O-GlcNAcylation. However, it is largely unknown which component(s) of the unfolded protein response (UPR) is directly regulated by O-GlcNAcylation. In this study, eukaryotic translation initiation factor 2α (eIF2α), a major branch of the UPR, was O-GlcNAcylated at Ser 219, Thr 239, and Thr 241. Upon ER stress, eIF2α is phosphorylated at Ser 51 by phosphorylated PKR-like ER kinase and this inhibits global translation initiation, except for that of specific mRNAs, including activating transcription factor 4, that induce stress-responsive genes such as C/EBP homologous protein (CHOP). Hyper-O-GlcNAcylation induced by O-GlcNAcase inhibitor (thiamet-G) treatment or O-GlcNAc transferase (OGT) overexpression hindered phosphorylation of eIF2α at Ser 51. The level of O-GlcNAcylation of eIF2α was changed by dithiothreitol treatment dependent on its phosphorylation at Ser 51. Point mutation of the O-GlcNAcylation sites of eIF2α increased its phosphorylation at Ser 51 and CHOP expression and resulted in increased apoptosis upon ER stress. These results suggest that O-GlcNAcylation of eIF2α affects its phosphorylation at Ser 51 and influences CHOP-mediated cell death. This O-GlcNAcylation of eIF2α was reproduced in thiamet-G-injected mouse liver. In conclusion, proper regulation of O-GlcNAcylation and phosphorylation of eIF2α is important to maintain cellular homeostasis upon ER stress.

Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation.

Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans.